Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Abstract

Hepatitis delta virus (HDV) is a unique human pathogen that increases the severity of liver disease in those infected with its helper virus, hepatitis B virus (HBV). The ϳ1,680-nucleotide (nt) single-stranded circular RNA genome assumes a characteristic unbranched rodlike structure in which ϳ70% of the nucleotides form Watson-Crick base pairs (1, 2). Genome replication occurs via a double-rolling-circle mechanism in which host RNA polymerase is redirected to synthesize genomic and antigenomic HDV RNAs, as well as the mRNA for hepatitis delta antigen (HDAg), the sole viral protein (reviewed in references 3 and 4). HDAg forms RNA-protein complexes (RNPs) with both genomic and antigenomic HDV RNAs, and these complexes play essential roles in this unique replication process (5-11). HDAg has been shown to transport HDV RNA to the nucleus, where replication occurs (12). In addition, HDAg interacts with RNA polymerase II (Pol II) (13,14); this interaction may recruit the polymerase to bound HDV RNA and has been proposed to effect changes in the polymerase that permit RNA-directed transcription (14-17). RNPs formed by HDAg and HDV RNA are also packaged into virions (18, 19); these complexes include a modified form of HDAg that interferes with RNA synthesis (20-24). Understanding how HDV RNPs function depends on knowing their structural features, which remains a critical goal.Several studies have attempted to identify regions of HDAg that directly contribute to binding HDV RNA and forming HDV RNPs, but a consistent picture has not yet emerged. In a widely cited model, two arginine-rich motifs (ARM I and ARM II) in the middle region of the protein are thought to form a bipartite RNA binding domain (8). This model is based largely on two in vitro binding studies using bacterially expressed HDAg fusion proteins. One of these studies showed that only fusion proteins containing the middle region of the protein, which includes the ARMs, could bind HDV RNA (25); no RNA binding activity was associated with the N-terminal 78 amino acids (aa). The other study showed loss of in vitro RNA binding activity when either of the ARMs, particularly ARM I, was disrupted by replacement of two basic residues with other amino acids (8). Both reports relied heavily on RNAprotein blots (Northwestern blots), which depend on the ability to remove bound detergent and properly refold proteins following electrophoresis and blotting. In contrast, other in vitro studies implicated the amino-terminal domain of HDAg in binding HDV . However, these analyses were limited by the use of small segments of the protein; Poisson et al. used small peptide fragments (28), and Huang and Wu and Wang et al. employed an N-terminal 88-aa region that bound equally well to HDV and non-HDV RNAs (26,27).Analyses of HDAg-HDV RNA interactions in cells have also yielded conflicting interpretations. Wang et al. (11) found that mutation of ARM I severely diminished the ability of HDAg to package the RNA into secreted particles, a result that could suggest a role for ARM I in ...

Hepatitis D Virus Replication

“…This binding required a delta RNA fragment of at least of 311 nucleotides (Defenbaugh et al 2009). With this in vitro system, it was recently found that the above-mentioned ARMs are not required (Daigh et al 2013). This supports a model in which a multimer of the delta antigen provides numerous sites at which contact is made with the HDV rod-like RNA.…”

Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex

“…Moreover, binding exhibits a remarkable length requirement of about 311 nt (20). We have also used HDAg-160 and a 395-nt segment of HDV RNA to show that the amino-terminal region of HDAg is closely associated with the RNA and that complex formation likely involves contact with many amino acids (25). Recent work has demonstrated that these results are not artifacts due to the use of a C-terminally truncated protein or the N-terminal His 6 tag: both HDAg-160 with an Nterminal 3ϫ-FLAG epitope tag instead of the His 6 tag and a His 6 -tagged, full-length protein from another HDV genotype exhibited the same RNA length and secondary-structure binding specificities as HDAg-160 (B. Griffin and J.…”

“…Native his-tagged HDAg-160 was expressed and purified as previously described (20,25). Protein purity and concentration were determined by Coomassie blue staining of proteins electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and by UV absorbance, both in comparison to bovine serum albumin (BSA) standards as previously described (20,25).…”

“…Electrophoretic mobility shift assays were performed as previously described (25). Mutated and substituted RNAs were always compared to a wild-type or unmodified RNA analyzed at the same time with the same protein preparation to control for any variations in binding activity in different protein preparations.…”

“…Alternatively, the presence of positively charged amino acids on the outer edges of the structure led to the suggestion that wrapping of the RNA around the protein could also be possible (22). Although the region crystallized appears to play a major role in RNA binding, other regions of the protein also contribute to binding specificity (25).…”